MILK OLIGOSACCHARIDE PROFILES BY REVERSED-PHASE HPLC OF THEIR PERBENZOYLATED DERIVATIVES

Human milk is rich in oligosaccharides, some of which inhibit toxins a nd pathogens involved in diseases of infants, To investigate qualitati ve and quantitative individual variation of human milk oligosaccharide s, a sensitive method for routine identification and quantification of intact milk oligosaccharides was developed and applied to milk sample s from 50 donors. The isolated, reduced neutral oligosaccharide fracti ons were perbenzoylated, resolved by reversed-phase HPLC, and detected at 229 nm. This method resolves most structural isomers and does not require stringent removal of lactose. Peaks were detected at the low n anogram (pmol) level and peak areas were linear from 1 to 1000 mu g fo r a standard oligosaccharide. Oligosaccharide samples equivalent to 1 mu l of human milk give optimum chromatographic separation and resolut ion. The method gives quantitative results comparable to those obtaine d with classic total sugar analyses, and has an average coefficient of variation of 13%, The 12 major peaks in human milk coeluted with auth entic oligosaccharide standards ranging from tri-to octasaccharides, a nd their identities were confirmed by mass spectrometry. Significant i ndividual variation exists in oligosaccharide profiles; almost 70% of samples contained 2'-fucosyllactose and lacto-N-fucopentaose I as the major oligosaccharides; for the remainder, the major oligosaccharides were 3-fucosyllactose and lacto-N-fucopentaose-II or lacto-N-fucopenta ose-III. This method can be used to investigate the extent and biologi cal significance of oligosaccharide variation in human milk. (C) 1997 Academic Press.